1. 156
Pak. j. life soc. Sci. (2010), 8(2): 156-158
RNA Extraction of Peste Des Petits Ruminants Virus (PPRV) from Clinical
Samples using Tri-Reagent and Acid Guanidinium Thiocyanate–Phenol–
Chloroform Methods
Samina Ashiq, Zulkifal Hussain, Muhammad Abubakar1
, Shamim Saleha, Muhammad Javed
Arshed2
and Qurban Ali2
Department of Microbiology, Kohat University of Science and Technology, Kohat, Pakistan
1
PARC Institute of Advance Studies in Agriculture (PIASA), NARC, Park Road, Islamabad, Pakistan
2
National Veterinary Laboratory, Park Road, Islamabad, Pakistan
Abstract
Peste des petits ruminants (PPR) is an acute and
highly contagious viral disease of small ruminants
such as goats and sheep. It causes economic losses
of Rs. 20.5 billion annually in Pakistan. The
diagnosis of PPR infection in sheep and goats
populations can be synergistically strengthened by
detection of antigen in clinical samples of
susceptible population. In present study, two RNA
extraction methods, i.e., Tri-reagent and Acid
guanidinium thiocyanate–phenol–chloroform
(AGPC) method were investigated for the PPR
virus antigen detection. Both Tri-reagent and Acid
guanidinium thiocyanate–phenol–chloroform
methods used for RNA extraction of PPRV have
equal importance. The RNA of PPRV was
extracted from ten tissue samples that were found
positive by Immuno-capture ELISA. RNA
extracted from these samples was quantified by
spectrophotometric analysis. The rapid detection
by such suitable and appropriate methods of
nucleic acid detection of PPR virus in infected
animals will help in early diagnosis of infection
and subsequent control of the PPR disease in
Pakistan.
Keywords: RNA extraction, Peste des petits
ruminants (PPR), Tri-reagent and AGPC methods.
Introduction
Peste des petits ruminants (PPR) is an acute and
highly contagious viral disease of small ruminants
such as goats and sheep. Abubakar et al., (2008) have
reported dramatic consequences with morbidity of
80-90% and mortality between 50 and 80% due to
infection of PPR virus in small ruminants. PPR is
endemic in Pakistan and has been reported from all
the four provinces. In Pakistan, it causes economic
losses of Rs. 20.5 billion annually. Clinical findings
are fever, anorexia, ocular and nasal discharges, sores
in mouth, diarrhea and pneumonia. The main routes
of PPR virus (PPRV) transmission are oral and
respiratory tracts and oral, nasal and ocular
excretions are the key sources (Couacy-Hymann et
al., 2009).
PPR virus (PPRV) is classified as a member of the
genus Morbillivirus in the family Paramyxoviridae. It
has single-strand negative sense RNA genome of
~16kb (15,948 nucleotides) in length that encodes
eight proteins including six structural proteins
namely: nucleoprotein (N), phosphoprotein (P),
matrix protein (M), fusion protein (F),
haemagglutinin protein (H) and large polymerase
protein (L), and two nonstructural proteins V and C
(Chard et al., 2008).
The small ruminants infected with PPR are routinely
diagnosed on the basis of clinical examination, gross
pathology, histologic findings and laboratory
confirmation. A number of serological and molecular
diagnostic tests are used for the detection of PPR
virus, including isolation on cell culture, agar gel
immunodiffusion (AGID), haemagglutination (HA)
test, haemagglutination inhibition (HI), competitive
enzyme-linked immunosorbent assay (c-ELISA),
immunocapture enzyme-linked immunosorbent assay
(IC-ELISA) and reverse transcriptase polymerase
chain reaction (RT-PCR) (Nussieba et al., 2008;
Forsyth and Barrett, 1995; Libeau et al.,1994).
The main objective of the study was to evaluate two
RNA extraction methods, i.e., Tri-reagent and Acid
guanidinium thiocyanate–phenol–chloroform method
for the PPR virus antigen detection.
Materials and Methods
A total of ten different tissues samples (lung, liver,
spleen, heart and lymph nodes) were collected from
PPR suspected outbreaks in sheep and goats from
different locations of Pakistan (Rawalpindi,
Faisalabad and Lahore).
These samples were tested for antigen detection of
PPRV using Immuno-capture ELISA. IC-ELISA was
Pakistan Journal of
Life and Social Sciences
Corresponding Author: Muhammad Abubakar
PARC Institute of Advance Studies in Agriculture
(PIASA), NARC, Park Road, Islamabad, Pakistan
Email: hayee42@yahoo.com

2. Peste Des Petits Ruminants Virus (PPRV) from Clinical Samples
157
performed using the kits made from BDSL (Pirbright,
UK) as described by Libeau et al., (1994). RNA of
PPRV was extracted from the positive samples by
Tri-reagent and Acid guanidinium thiocyanate–
phenol–chloroform (AGPC) methods.
RNA Extraction
(i) Trireagent Method:
Five samples that were found positive for PPR virus
by IC-ELISA were further processed for isolation of
RNA by Trireagent (Molecular Research Center
Inc.).
(ii) Acid guanidinium thiocyanate–phenol–
chloroform (AGPC) Method:
The same five samples were used for isolation of
RNA by AGPC method as described by
Chomczynski and Sacchi (2006).
RNA Quantification by Spectrophotometry
RNA was quantified by spectrophotometric analysis
using the convention that one absorbance unit at 260
nm wavelength equals 40 μg RNA per ml. The ultra
violet absorbance was checked at 260 and 280 nm for
determination of RNA concentration and purity.
Purity of RNA was judged on the basis of optical
density ratio at 260:280 nm. The following formula
was used to determine RNA concentration of the
original sample:
Concentration of RNA (μg/μl) =
A260 x Dilution factor x 40
1000
Results
The RNA of PPRV was extracted from five samples
that were found positive by IC-ELISA, by Trireagent
and AGPC methods. Same samples were extracted by
Tri-reagent and AGPC methods. RNA extracted from
all samples was quantified by spectrophotometric
analysis. Purity of the extracted RNA (i.e. ratio 1.7-
2.0) was judged on the basis of optical density ratio at
260:280 nm as shown in tables 1 and 2.
Both Tri-reagent and AGPC methods showed equal
compassion in RNA extraction although the AGPC is
standardized in the laboratory while the Tri-reagent is
the commercially standard method. All the samples
were then confirmed for the F-gene of PPRV using
Polymerase Chain Reaction (PCR) (Forsyth and
Barrett, 1995).
Discussion
In this study, the PPRV antigen detection and RNA
extraction was investigated and compared in clinical
samples of small ruminants. The RNA of PPRV was
extracted by Tri-reagent and AGPC methods. Both
Tri-reagent and AGPC methods are recommended as
initial steps in molecular diagnosis of PPRV in
various studies. Farooq et al. (2008) also utilized
AGPC method for RNA extraction in a study on
molecular based diagnosis of Rinderpest and Peste
Des Petits Ruminants Virus in Pakistan.
Balamurugan et al., (2006) utilized Tri-reagent
method in a study while using one-step multiplex
RT-PCR Assay for the detection of Peste des petits
ruminants virus in clinical samples. Results of this
study showed that both RNA extraction methods
Table 1 Optical density ratio at 260:280 nm of RNA samples extracted by Tri-reagent method
Sr # Samples
Dilution factor (500) Dilution factor (200)
RNA µg/µl
260/ 280
RNA µg/µl
260/ 280260nm 280nm 260nm 280nm
1. Sample 1 1.68 1.72 0.97 0.728 0.68 1.07
2. Sample 2 3.38 2.7 1.25 2.656 1.528 1.73
3. Sample 3 1.74 1.64 1.04 0.688 0.648 1.06
4. Sample 4 1.76 1.66 1.06 0.712 0.664 1.07
5. Sample 5 1.78 1.68 1.05 0.64 0.616 1.03
Table 2 Optical density ratio at 260:280 nm of RNA samples extracted by AGPC method
Sr # Samples
Dilution factor (500) Dilution factor (200)
RNA µg/µl
260/280
RNA µg/µl
260/280260 280 260 280
1. Sample 1 0.188 1.74 1.08 0.608 0.576 1.05
2. Sample 2 1.86 1.88 0.98 0.88 0.8 1.1
3. Sample 3 1.76 1.76 1.00 0.832 0.752 1.106
4. Sample 4 1.48 1.46 1.01 0.776 0.72 1.07
5. Sample 5 1.26 1.32 0.95 0.648 0.6 1.08

3. Ashiq et al
158
have equal importance and can be utilized in
extraction of RNA of PPRV. In this study we carried
out the initial steps for molecular diagnosis of PPRV
by Trireagent and AGPC methods and efficiency of
both methods was also compared.
IC-ELISA is used as standard since it has the best
sensitivity and specificity and can be utilized for
samples which are not kept under ideal conditions.
RT-PCR is considered the most precise and sensitive
technique for diagnosis in clinical samples of PPRV
as compared to IC-ELISA, AGID and HA for
increased sensitivity and reduced false positivity
(Farooq et al., 2008).
The diagnosis of PPR infection in sheep and goats
populations can be synergistically strengthened with
detection of antigen in clinical samples of susceptible
population. Thus rapid detection by suitable and
appropriate methods of antigen and nucleic acid
detection of PPRV in infected animals will help in
early diagnosis of infection and subsequently control
of the PPR disease in Pakistan.
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